تشخيص زودرس ليشمانيوز احشايي با استفاده از خون محيطي و روش واكنش زنجيره اي پليمراز

Early Diagnosis of Visceral Leishmaniasis by Using Polymerase Chain Reaction Method on Whole Blood

Visceral leishmaniasis or kala-azar, is a worldwide disseminated intracellular infection of the liver, spleen and bone marrow. Mortality and morbidity of visceral leishmaniasis is about 15% even in case of treatment. Determination of antilesihmania antibody in serum of patients using indirect fluorescent technique (IFA) and detection of leishman bodies in bone marrow smear are presently used for definitive diagnosis of visceral leishmaniasis in Iran. Bone marrow puncture is an invasive method and inconvenient for patients. Moreover, because of some technical problems, the sensitivity of the test is quit low. The ability to diagnosis visceral leishmaniasis using easily obtained material, such as patientsʹ blood, would be a major advance. The aim of this study was to determine the ability of PCR method for detection of Leishmania DNA in peripheral blood of patients with visceral leishmaniasis. The results were then compared with IFA method. A total of 67 whole blood and sera were collected from patients suspected of having visceral leishmaniasis. Polymerase chain reaction on whole blood and indirect fluorescent antibody on sera were applied for these patients. DNA extraction was carried out on whole blood by using lysis buffer and boiling method. Leishmania-specific primers (13A and 138) were used to amplify 120 base pair fragment of Leishmania kinetoplast DNA (kDNA) minicircles. Microscopically examination of smears of bone marrow was carried out for the patients during hospitalization. In 27 patients, the diagnosis of visceral leishmaniasis was made by clinical symptoms, laboratory data, response to treatments and final clinical diagnosis. 23 of 27 patients with confirmed visceral leishmaniasis had positive PCR (85.2% sensitivity). None of 40 cases with others infections had PCR- positive (100% specificity). Sensitivity and specificity of IFA was 63% and 82.5%, respectively. There was statistically difference between specificity of these methods (P value < 0.01). These results demonstrate the high specificity and sensitivity of the non-invasive PCR. The patients with early stage of visceral leishmaniasis were detected by this method as well. Therefore, PCR is a valuable method for the diagnosis of visceral leishmaniasis in compare of 1FA.